DNA-DNA hybridization is a molecular biology technique that measures the degree of sequence similarity between deoxyribonucleic acid (DNA) polymers (polynucleotides). The underlying principle is that the building blocks of the DNA polymer, i.e., nucleotides, include specific nitrogen-containing nucleobases (guanine “G,” adenine “A,” thymine “T,” and cytosine “C”) capable of pairing up with complementary nucleobases (A with T and C with G) to form hydrogen bonds (two (2) between A-T and three (3) between C-G). Therefore, DNA moieties with complementary sequences have an affinity to bind (hybridize) to one another and DNA dimers (double stranded DNA structures). The thermodynamics characteristics hybridization depends predominately on the total number, and strength of hydrogen bonds formed between the DNA moieties; a quantity which is a function of multiple parameters such as complementary nucleobase (base) stretches, non-complementary gaps, and the concentration and variety of anions and cations in the environment.
The thermodynamic characteristic of DNA-DNA-hybridization is a powerful tool to infer sequence information regarding the participating moieties. The “gold standard” method to extract such information is melt curve analysis (MCA) which detects the dissociation-characteristics of double-stranded and hybridized DNA dimers during a gradual heating process. As temperature is raised, the DNA-DNA complex (assembled through multiple hydrogen bonds) becomes less stable and the strands begin to dissociate. Thus, by monitoring the concentration of hybridized complexes versus temperature, one can evaluate the stability of the complex as a function of temperature and correlate it to alterations within the target sequence (and hydrogen bonds) and, for example, identify single-nucleotide polymorphisms (“SNPs”) or insertions/deletions (“indels”).